Small-molecule Ro-08-2750 interacts with many RNA-binding proteins and elicits MUSASHI2-independent phenotypes.

RNA-binding proteins (RBPs) are key regulators of gene expression. Small molecules targeting these RBP-RNA interactions are a rapidly emerging class of therapeutics for treating a variety of diseases. Ro-08-2750 (Ro) is a small molecule identified as a competitive inhibitor of Musashi (MSI)-RNA interactions. Here, we show that multiple Ro-dependent cellular phenotypes, specifically adrenocortical steroid production and cell viability, are Musashi-2 (MSI2)-independent. Using an unbiased proteome-wide approach, we discovered Ro broadly interacts with RBPs, many containing RRM domains. To confirm this finding, we leveraged the large-scale ENCODE data to identify a subset of RBPs whose depletion phenocopies Ro inhibition, indicating Ro is a promiscuous inhibitor of multiple RBPs. Consistent with broad disruption of ribonucleoprotein complexes, Ro treatment leads to stress granule formation. This strategy represents a generalizable framework for validating the specificity and identifying targets of RBP inhibitors in a cellular context.

RNA-binding proteins regulate aldosterone homeostasis in human steroidogenic cells.

Angiotensin II (AngII) stimulates adrenocortical cells to produce aldosterone, a master regulator of blood pressure. Despite extensive characterization of the transcriptional and enzymatic control of adrenocortical steroidogenesis, there are still major gaps in the precise regulation of AII-induced gene expression kinetics. Specifically, we do not know the regulatory contribution of RNA-binding proteins (RBPs) and RNA decay, which can control the timing of stimulus-induced gene expression. To investigate this question, we performed a high-resolution RNA-seq time course of the AngII stimulation response and 4-thiouridine pulse labeling in a steroidogenic human cell line (H295R). We identified twelve temporally distinct gene expression responses that contained mRNA encoding proteins known to be important for various steps of aldosterone production, such as cAMP signaling components and steroidogenic enzymes. AngII response kinetics for many of these mRNAs revealed a coordinated increase in both synthesis and decay. These findings were validated in primary human adrenocortical cells stimulated ex vivo with AngII. Using a candidate screen, we identified a subset of RNA-binding protein and RNA decay factors that activate or repress AngII-stimulated aldosterone production. Among the repressors of aldosterone were BTG2, which promotes deadenylation and global RNA decay. BTG2 was induced in response to AngII stimulation and promoted the repression of mRNAs encoding pro-steroidogenic factors indicating the existence of an incoherent feedforward loop controlling aldosterone homeostasis. These data support a model in which coordinated increases in transcription and decay facilitate the major transcriptomic changes required to implement a pro-steroidogenic expression program that actively resolved to prevent aldosterone overproduction.

In Situ Spatial Reconstruction of Distinct Normal and Pathological Cell Populations Within the Human Adrenal Gland.

The human adrenal gland consists of concentrically organized, functionally distinct regions responsible for hormone production. Dysregulation of adrenocortical cell differentiation alters the proportion and organization of the functional zones of the adrenal cortex leading to disease. Current models of adrenocortical cell differentiation are based on mouse studies, but there are known organizational and functional differences between human and mouse adrenal glands. This study aimed to investigate the centripetal differentiation model in the human adrenal cortex and characterize aldosterone-producing micronodules (APMs) to better understand adrenal diseases such as primary aldosteronism. We applied spatially resolved in situ transcriptomics to human adrenal tissue sections from 2 individuals and identified distinct cell populations and their positional relationships. The results supported the centripetal differentiation model in humans, with cells progressing from the outer capsule to the zona glomerulosa, zona fasciculata, and zona reticularis. Additionally, we characterized 2 APMs in a 72-year-old woman. Comparison with earlier APM transcriptomes indicated a subset of core genes, but also heterogeneity between APMs. The findings contribute to our understanding of normal and pathological cellular differentiation in the human adrenal cortex.

Evolution of the GST omega gene family in 12 Drosophila species.

Gene families provide a unique system to study the evolutionary relationships between related genes both within and between organisms. We can ascertain whether members of a gene family in different species are orthologs or paralogs. We may also search for evidence that may explain why duplicate genes are present. The availability of genome sequences for 12 Drosophila species allows us to address these questions with respect to the evolution of one gene family, the glutathione S transferase (GST) omega class. This gene family is of particular interest because of its relationship with human disease and its presence in a wide range of species.

Luteinizing hormone-releasing hormone I (LHRH-I) and its metabolite in peripheral tissues.

Luteinizing hormone-releasing hormone (LHRH) was first isolated in the mammalian hypothalamus and shown to be the primary regulator of the reproductive system through its initiation of pituitary gonadotropin release. Since its discovery, this form of LHRH (LHRH-I) has been shown to be one of many structural variants with a variety of roles in both the brain and peripheral tissues. Enormous interest has been focused on LHRH-I and LHRH-II and their cognate receptors as targets for designing therapies to treat cancers of the reproductive system. LHRH-I is processed by a zinc metalloendopeptidase EC 3.4.24.15 (EP24.15) that cleaves the hormone at the fifth and sixth bond of the decapeptide (Tyr(5)-Gly(6)) to form LHRH-(1-5). We have previously reported that the autoregulation of LHRH gene expression can also be mediated by its processed peptide, LHRH-(1-5). Furthermore, LHRH-(1-5) has also been shown to be involved in cell proliferation. This review will focus on the possible roles of LHRH and its processed peptide, LHRH-(1-5), in non-hypothalamic tissues.

A processed metabolite of luteinizing hormone-releasing hormone has proliferative effects in endometrial cells.

OBJECTIVE: The purpose of this study is to determine the possible role of the processed peptide of LHRH, LHRH-(1-5), in regulating growth of endometrial cancer cells. STUDY DESIGN: An endometrial cancer cell line, the Ishikawa cell line, was cultured under standard conditions and treated in a dose-dependent manner with 1 of 2 hormones, LHRH and LHRH-(1-5) to determine the ability of these peptides to regulate cellular growth. A tetrazolium-based assay was used to determine the effect these peptides have on cell proliferation. Furthermore, enzyme-linked immunosorbent assay (ELISA)-based assays were used to determine the expression of caspase-3/7 and pERK-1/2. Statistical analyses were conducted using an analysis of variance followed by Fisher LSD as the post-hoc test. RESULTS: The results show that LHRH is anti-proliferative whereas LHRH-(1-5) is proliferative on the cells. Furthermore, LHRH-(1-5) decreased caspase-3/7 and pERK1/2 expression. CONCLUSION: This is the first time LHRH-(1-5) is shown to have proliferative effects on cells.

The perceived urgency of speech warnings: semantics versus acoustics.

The relationship between the semantics of words and the acoustics of the way they are spoken is explored. Actors spoke warning signal words in an urgent, nonurgent and monotone style, and participants rated the urgency of the words. Results showed effects for signal word and style of presentation. Acoustic analysis showed that the urgent words were spoken at higher frequency with a broader pitch range and were louder than the nonurgent or monotone words. These acoustic differences were used to synthesize artificial versions of signal words in urgent and nonurgent formats. The urgent words were rated as more urgent than the nonurgent words, a finding attributable to their differing acoustics. Within each speaking style the words were acoustically the same, yet effects for signal word were found, suggesting that semantics is also important in urgency perception. This research has implications for the design and implementation of speech warning systems, particularly those in which urgency mapping is required.